A single nucleotide polymorphism is a difference among the individuals existing on a genomic DNA, which may cause differences in various diseases and various phenotypes in human and the like. Therefore, the SNP is utilized in analysis of genetic diseases, discriminations between the individuals, and the like.
At present, real-time PCR method used in the detection of a single nucleotide polymorphism (SNP) include, for example, Taq Man (Registered Trademark) method and SYBR (Registered Trademark) Green method. The Taq Man (Registered Trademark) method is a highly sensitive method: however, the design and synthesis of Taq Man (Registered Trademark) probe used in the detection are complicated, thereby making the detection cost high. In addition, the SYBR (Registered Trademark) Green method is a convenient method utilizing that fluorescent intensity increases by the binding of a double-stranded DNA; however, formation of double strands by nonspecific amplification would also be detected as being “positive,” the detection error is large, so that there is a problem of optimizing the primers used for increases allelic specificity. As the primers in these methods, fairly correct primers to some degree are designed using designing software, and thereafter the primer sequences and conditions for PCR are optimized so that the allelic specificity would be the highest. However, optimization on individual genomes would be necessitated, so much works are needed to find optimal conditions.
By contrast, the present inventors have reported a hairpin primer PCR (HP-PCR) method utilizing the fluorescent properties that compounds containing a naphthyridine ring specifically bind to bulge structures, thereby shifting from a wavelength of maximum absorbance before binding, and that the fluorescent intensity fluctuates depending upon the kinds of nucleotide residues pairing with nucleotides adjoining the bulge nucleotide (see Patent Publication 1). Concretely, first, primers having a hairpin structure in which a bulge region to which the compounds containing a naphthyridine ring specifically bind is introduced at a 5′-terminal are prepared, and next, the subject nucleic acids are hybridized by PCR to form a duplex containing a bulge structure. The naphthyridine ring-containing compounds are added thereto and bound to the bulge structure, thereby shifting the wavelength of maximum absorbance, which is observed as fluctuations of fluorescent intensities.